Development of the complex three-dimensional structure of the inner ear requires precise coordination of cell fate specification, proliferation, survival, and morphology. Even subtle failures during one developmental process can have catastrophic consequences. Indeed, many human deafness and balance disorders are caused by defects in the development of the auditory system, ranging from a complete loss of the semicircular canals or cochlea to auditory circuitry deficits that interfere with sound perception. The long term goal of our work is to understand how genetic mutations lead to changes in the structure and function of the inner ear, ultimately resulting in deafness and vestibular dysfunction. One of the enduring mysteries in our field is how three hollow semicircular canals are formed from an initially simple sphere of epithelium. A key step in this process is a fusion event that eliminates cells in the center of a canal pouch while leaving the surrounding canal rim intact. Although Netrin1 (Ntn1) and FGF proteins are involved, very little is known about how these secreted proteins interact to determine when and where fusion occurs. We have defined a novel pathway for canal morphogenesis. We found that the Ig superfamily protein Lrig3 participates in reciprocal interactions with Ntn1 that ultimately restrict fusion to the center of the pouch. Lrig3 is a poorly characterized transmembrane protein with leucine rich repeats (LRR) and immunoglobulin (Ig) domains in its extracellular domain. While Lrig proteins are able to antagonize receptor tyrosine kinase (RTK) signaling in some contexts, how this occurs at the molecular level is not known. Since FGF receptors are RTKs and can bind to Lrig3, we hypothesize that Lrig3 provides a link between FGF signaling and Netrin1 during canal morphogenesis. Further, we propose that all three Lrig proteins share a common ability to fine tune FGF and other signaling pathways elsewhere in the developing and mature inner ear. In support of this idea, Lrig1 and Lrig2 are also expressed in the inner ear and are required for hearing. In this study, we will complement phenotypic analysis in mice and chicks with biochemical investigations of Lrig, FGFR, and Ntn proteins in order to gain insights into the cellular and molecular mechanisms that govern inner ear development. Specifically, we will ask 1) Does Lrig3 act through an FGF signaling pathway to regulate Ntn1 expression? 2) What are the subsequent consequences of Ntn1 activity at the cellular level? and 3) Do Lrig proteins share a role in receptor trafficking that influences other aspects of inner ear development? These studies will improve our knowledge of the cellular and molecular basis of ear morphogenesis and elucidate the functions of Ntn and Lrig proteins, which have been implicated in tumor formation and invasion in humans. PUBLIC HEALTH RELEVANCE: Many human deafness and balance disorders are caused by genetic mutations that disrupt development of the inner ear, an intricate structure that houses specialized cells for the detection of sound or motion. An effective approach toward understanding the origins of human inner ear defects is to identify genes that are required for inner ear development in animal models. A thorough knowledge of how these genes direct formation of the inner ear in mice will facilitate the identification of deafness genes in humans, improve detection and diagnosis of human deafness and balance disorders, and uncover new targets for therapeutic intervention.